Normal serum contains a number of lipoprotein particles which are characterized according to their density, namely, chylomicrons, VLDL, LDL and HDL. They are composed of free and esterified cholesterol, triglycerides, phospholipids, several other minor lipid components, and protein. Low density lipoprotein (LDL) transports lipid soluble materials to the cells in the body, while high density lipoprotein (HDL) transports these materials to the liver for elimination. Normally, these lipoproteins are in balance, ensuring proper delivery and removal of lipid soluble materials. Abnormally low HDL can cause a number of diseased states as well as constitute a secondary complication in others.
Under normal conditions, a natural HDL particle is a solid with its surface covered by a phospholipid bilayer that encloses a hydrophobic core. Apolipoprotein A-I and A-II attach to the surface by interaction of the hydrophobic face of their alpha helical domains. In its nascent or newly secreted form the particle is disk-shaped and accepts free cholesterol into its bilayer. Cholesterol is esterified by the action of lecithin:cholesterol acyltransferase (LCAT) and is moved into the center of the disk. The movement of cholesterol ester to the center is the result of space limitations within the bilayer. The HDL particle "inflates" to a spheroidal particle as more and more cholesterol is esterified and moved to the center. Cholesterol ester and other water insoluble lipids which collect in the "inflated core" of the HDL are then cleared by the liver.
Jonas, et al., Meth. Enzym. 128A: 553-582 (1986) have produced a wide variety of reconstituted particles resembling HDL. The technique involves the isolation and delipidation of HDL by standard methods (Hatch, et al., Adv. Lip. Res. 6: 1-68 (1968); 576-588 (1971) to obtain apo-HDL proteins. The apoplipoproteins are fractionated and reconstituted with phospholipid and with or without cholesterol using detergent dialysis.
Matz, et al., J. Biol. Chem 257(8): 4535-4540 (1982) describe a micelle of phosphatidylcholine, with apolipoprotein Al. Various ratios of the two components are described, and it is suggested that the described method can be used to make other micelles. It is suggested as well to use the micelles as an enzyme substrate, or as a model for the HDL molecule. This paper does not, however discuss application of the micelles to cholesterol removal, nor does it give any suggestions as to diagnostic or therapeutic use.
Williams, et al., Biochem & Biophys. Acta 875: 183-194 (1986) teach phospholipid liposomes introduced to plasma which pick up apolipoproteins and cholesterol. Liposomes are disclosed, which pick up apolipoprotein in vivo, as well as cholesterol, and it is suggested that the uptake of cholesterol is enhanced in phospholipid liposomes which have interacted with, and picked up apolipoproteins.
Williams, et al., Persp. Biol. & Med. 27(3): 417-431 (1984) discuss lecithin liposomes as removing cholesterol. The paper summarizes earlier work showing that liposomes which contain apolipoproteins remove cholesterol from cells in vitro more effectively than liposomes which do not contain it. They do not discuss in vivo use of apolipoprotein containing liposomes or micelles, and counsel caution in any in vivo work with liposomes.
It is important to note that there is a clear and significant difference between the particles of the present invention, and the liposomes and micelles described in the prior art. The latter involve a bilayer structure of lipid containing molecules, surrounding an internal space. The construction of liposomes and micelles precludes filling the internal space, however, and any molecular uptake is limited to the space defined between the two lipid layers. As a result, there is much less volume available for pick up and discharge of materials such as cholesterol and other lipid soluble materials than there is for the particles of this invention, which expand in a fashion similar to a balloon, with interior space filling with the material of choice.
The present invention involves the production of reconstituted particles with and without free cholesterol in the bilayer using detergent dialysis These particles appear when viewed by negative staining transmission electron microscopy (TEM) as discoidal and are transformed to spheroidal particles upon exposure to LCAT. They retain their ability to act as substrates for LCAT and are transformed to spheroidal particles in a fashion similar to natural HDL. When reconstituted particles containing 10 percent mole fraction of tritiated cholesterol in the bilayer are exposed to LCAT for 12 hours, almost complete conversion of free cholesterol to cholesterol ester is achieved as determined by thin layer chromatography (TLC).
In summary, synthetic HDL particles are structural and functional analogs of natural HDL particles in that they (1) resemble nascent HDL particles when visualized under negative staining TEM, (2) are substrates for LCAT and (3) function as cholesterol acceptors which deplete cholesterol and other lipid soluble toxins from cells, such as human cells.